Method for the Production of Suitable Dna Constructs for Specific Inhibition of Gene Expression by Rna Interference

ABSTRACT

The invention relates to a method for the production of vectors which, following transfection thereof in eukaryotic cells, are suitable for targeted inhibition of the formation of defined proteins therein by RNA interference. The method for the production of such vectors does not include any PCR steps. It is a three-step procedure in a single reaction vessel and can be carried out within a few hours. Thus, a method is provided which allows very easy testing of a wide variety of siRNA sequences for their functionality within a very short time. Screening processes utilizing the rapid and uncomplicated production of vectors with the aid of said kit can be performed in a cost- and time-saving manner. Another advantage of vectors thus produced is their small size which, among other things, facilitates transfection.

The invention relates to a method for the production of vectors which,following transfection thereof in eukaryotic cells, are suitable fortargeted inhibition of formation of defined proteins therein by RNAinterference.

One recently detected way of inhibiting gene expression is based on theproduction of double-stranded RNA molecules. Using such double-strandedRNA (dsRNA), targeted switching off of single genes is possible in ahighly effective manner and more rapidly compared to any other method,without impeding protein formation of neighboring genes. The basicprinciple is referred to as RNA interference, abbreviated as RNAi, andthe dsRNA sequence responsible for this phenomenon as siRNA (smallinterference RNA).

The siRNA does not prevent reading of the gene, but rather switches on acellular mechanism causing degradation of the mRNAs read from the gene,thus pre-venting formation of the corresponding protein(post-transcriptional gene silencing).

Such targeted mRNA degradation is triggered by short siRNA molecules19-23 RNA bases in length which are homologous to the target mRNA whosetrans-formation into a protein is to be prevented. The siRNA moleculescombine with specific endoribonucleases to form a cellular RNA proteincomplex referred to as RISC (RNA-induced silencing complex). Duringformation of these complexes, the two RNA strands undergo dissociation,thereby forming so-called activated RISCs, each one including a singlestrand of the siRNA molecule. Activated RISCs including the antisensestrand which is complementary to the target mRNA bind thereto, and theendoribonuclease of the RNA-protein complex subsequently provides forsequence-specific mRNA degradation.

The siRNA can be generated in the cell by way of experiment or can beincorporated by introduction from the outside. On the one hand, this canbe done via synthetically produced siRNA molecules which can beadministered both in vitro and in vivo.

However, this method has technical limitations. In addition to thegeneral instability of synthetic siRNA both in a medium and in a cell,inhibition by means of a synthetic siRNA is, in principle, only possiblein a transient fashion, and transfection of a large number of cells(e.g. neuronal cells) is extremely inefficient. For this reason, studiesbased on the transfection with synthetic siRNA are generally restrictedin time to 1-5 days and in terms of a specific cell type. Furthermore,the high production cost and long production time are disadvantageous.

On the other hand, siRNA can be produced in the cell by vectors, saidvectors being viral or plasmid-based vectors leading to formation ofsiRNA sequences later inside the cell by expression. The advantages overtransfection with synthetic siRNA lie in a more stable and optionallyregulated transcription of the corresponding siRNA sequence.

However, in addition to low transfection efficiency, plasmid-basedvectors involve a complex production process. Thus, selection of stableclones is necessary, for example. During this process, usually being alengthy one, which can take weeks or even months, a number of potentialproblems inherent to cloning experiments frequently arise. Checking theproducts requires sequencing which likewise is labor- andcost-intensive.

Further, plasmid-based vectors include antibiotic resistance genesrequired for the selection thereof. For this reason, such vectors arenot suitable for use in living organisms. As a consequence of possiblerecombination with bacteria ubiquitous in the organism, there is a riskof increasing occurrence of antibiotic-resistant bacteria. Spreading ofantibiotic resistances represents a serious problem and isunjustifiable.

Viral vectors are capable of efficient and targeted transfection, forwhich reason they offer advantages compared to synthetic siRNA moleculesand plasmid-based vectors.

However, such viral vectors can be used in therapeutic applications onlywith reservations. Recombination of viral sequences with naturallyoccurring viruses represents an inherent safety risk in this case aswell because it must be feared that new, pathogenic hybrid viruses wouldbe formed. Moreover, the production thereof is also complex andcost-intensive.

Another way of producing vectors for siRNAs has been demonstrated by theAmbion Company on their internet site. The illustrated process avoidsthe above-mentioned drawbacks. Likewise, however, this productionprocess is time-consuming and quite imperfect as a result of a number ofnecessary amplification steps of the respective sequences by means ofPCR (polymerase chain reaction). It is very well possible that bothundesirable and unnoticed mutations are produced which are evenpotentiated by the PCR process. In this case as well, controlsequencings are required which prolong the production process andcontribute to increased cost.

In view of the above prior art, the object of the invention is toprovide a suitable method for the in vitro or in vivo synthesis of adefined siRNA sequence and a kit appropriate for this purpose.

Said object is accomplished by the characterizing features of claims 1and 11.

In the meaning of the invention, siRNA sequence is understood to be theRNA sequence that is read from the DNA construct produced according tothe invention. Hence, it is a singular RNA single strand being partiallyself-complementary.

In the meaning of the present invention the designation siRNA moleculeis used for an siRNA which is formed as a result of refolding and basepairing of a self-complementary siRNA sequence. Hence, an siRNA moleculeis a double-stranded RNA molecule in which the pairing strands arelinked on one side by a non-complementary single strand.

According to the invention, a method is provided which is characterizedby the following steps:

-   a) mixing a DNA double strand which includes a singular copy 19-23    bases in length of a gene sequence, once in 5′-3′ direction and once    in 3′-5′ direction, a sequence 8-12 bases in length of two single    strands being arranged between each 5′-3′ and 3′-5′ orientation of    the singular copy of the gene sequence, said single strands being    selected such that opposite bases are by no means complementary to    each other and the flanking double strand regions are thereby linked    to each other by two DNA single strands, said DNA double strand    having short protruding ends of single-stranded DNA at the ends    thereof,    -   with    -   hairpin loop-shaped oligodeoxynucleotides having short        protruding ends of single-stranded DNA at the ends thereof,    -   and    -   a promoter having short protruding ends of single-stranded DNA,        the single-stranded 5′ end of the promoter being capable of        pairing with one of the hairpin loop-shaped        oligodeoxynucleotides, and the single-stranded 3′ end of the        promoter being complementary to the single-stranded 5′ end of        the DNA double strand,    -   and    -   a termination signal for RNA polymerases with short protruding        ends of single-stranded DNA, the 5′ protrusion of the        termination signal being capable of specific pairing with the 3′        end of the DNA double strand, and the 3′ protrusion of the        termination signal being capable of specific pairing with a        hairpin loop-shaped oligodeoxynucleotide,-   b) subsequent ligation of the DNA fragments, and-   c) final purification of the vectors produced.

In a preferred embodiment, a method is provided wherein the promoter ispart of a bacterially amplifyable plasmid which, prior to mixing thecomponents in the first process step 1a), is cut with a restrictionendonuclease recognizing a restriction site flanking the promoter on theplasmid, which restriction site is not present on the molecule to beproduced.

According to the invention, it is also envisaged that in case of using apromoter as part of a bacterially amplifyable plasmid, the ligation stepis effected in the presence of the restriction endonuclease by means ofwhich the promoter has been excised from the plasmid.

In one embodiment the step of final purification is preceded bydigestion of the reaction mixture, using an exonuclease specific for 3′or 5′ DNA ends only.

In the method according to the invention, the DNA double strand added tothe mixture at the beginning may result from an annealing of a partiallyself-complementary oligodeoxynucleotide or of two complementaryoligodeoxynucleotides. It is also possible to effect annealing later inthe reaction mixture, so that merely single-stranded complementaryoligodeoxynucleotides are added at the beginning of the method accordingto the invention.

In a preferred embodiment the sequence of the oligodeoxynucleotides isselected in such a way that the resulting hairpins have the recognitionsequence for a restriction endonuclease in their double-stranded region.

The final purification of the vectors produced by means of the methodaccording to the invention is preferably effected using eitherchromatography or gel electrophoresis.

If the promoter is employed in the production process of the inventionas part of a bacterially amplifyable plasmid, the restrictionendonuclease by means of which the promoter can be excised from theplasmid is an enzyme from the group of class II restrictionendonucleases, preferably from the group of BbsI, BbvI, BbvlI, BpiI;BplI, BsaI, BsmAI, BsmBI, BsmFI, BspMI, Eam1104I, EarI, Eco31I, Esp3I,FokI, HgaI, SfaNI or isoschizomers thereof.

The present invention is also directed to a kit used to carry out themethod according to the invention, said kit including at least onepromoter, hairpin loop-shaped oligodeoxynucleotides, and enzymes. Theenzymes are ligases, restriction endonucleases, restrictionexonucleases, kinases and polymerases or selected combinations thereofin the form of an enzyme mix. In addition, depending on the particularembodiment, the kit may include means for performing the enzymaticreactions, as well as means for purifying the vectors produced. Thepromoter can be included in the kit as part of a plasmid from which itcan be excised using a suitable restriction endonuclease.

The present invention is also directed to a vector which is producedaccording to the method of the invention and which is capped by hairpinloop-shaped oligodeoxynucleotides having arranged therebetween apromoter at the 5′ end and a termination signal at the 3′ end of a DNAdouble strand, said DNA double strand including a singular copy 19-23bases in length of a gene sequence, once in 5′-3′ direction and once in3′-5′ direction, a sequence 8-12 bases in length of two single strandsbeing arranged between each 5′-3′ and 3′-5′ orientation of the singularcopy of the gene sequence, said single strands being selected such thatopposite bases are by no means complementary to each other and theflanking double strand regions are thereby linked to each other by twoDNA single strands. These expression cassettes are also referred to asminimalistic siRNA expression cassettes (MISECs).

The present invention differs from the well-known prior art in that arapid method is provided by means of which a vector is produced which isfree of plasmid or viral components and results in expression of siRNAsequences. The method for the production of such vectors does notinvolve any PCR steps, it is a three-step procedure and can be carriedout in a single reaction vessel within a few hours. Thus, a method isprovided which allows very easy testing of a wide variety of siRNAsequences for their functionality within a very short time. Screeningprocesses for suitable siRNA sequences, utilizing the rapid anduncomplicated production of vectors with the aid of said kit, can beperformed in a cost- and time-saving manner. Another advantage of thevectors thus produced is their small size which, among other things,facilitates transfection.

The siRNA sequences are single-stranded, comprising one sense strand andone antisense strand, each one comprising 19-23 nucleotides. The senseand antisense strands are separated by a short spacer region allowingsubsequent folding of the strands to form a double-stranded siRNAmolecule. This siRNA pairs with a target mRNA, resulting in degradationthereof by nucleases as described above.

The vector generated by means of said production process merelycomprises a suitable promoter sequence, the siRNA sequence to beexpressed, and a short termination sequence, and therefore does not bearany undesirable sequences of viral or plasmid origin. To protect fromdegradation by exonucleases, each end is covalently linked with a loopof single-stranded oligodinucleotides (ODN) so as to form a fullycovalently capped molecule.

In an alternative production process according to the invention the DNAsequences complementary to each other, not separated by single-strandedregions, are located in a single vector. Each of the complementarydouble-stranded sequences 19-23 bases in length (sense and antisense)are included in separate vectors which can be produced in an analogousfashion using the method according to the invention. Consequently, thevectors thus produced have the same structure as the vector includingthe sense-loop-antisense DNA strand between promoter and hairpin loop,but lack the single-stranded region.

In principle, any eukaryotic promoter sequence such as the CMV promoterof cytomegalovirus is suitable as promoter for transcription control. Itis preferred to use type III polymerase promoters such as H1 promoter,7SK promoter, as well as the human and murine U6 promoter. The promotersequence can be present on a suitable plasmid vector, from which it mustbe excised by means of restriction endonucleases at the beginning ofproduction, but it is also possible to add the promoter sequence to theprocess in the form of a previously isolated or synthetically producedsequence.

Any known DNA sequence resulting in termination of expression via RNApolymerases is possible as termination sequence. Separate addition ofthe termination sequence to the process according to the invention isnot necessary; instead, it can also be part of the double-strandedregion of a hairpin loop-shaped oligodeoxynucleotide or of the 3′ end ofthe partial DNA double strand which has two single strands in the centerthereof.

The siRNA sequence to be expressed is the sequence complementary to thetarget mRNA, which sequence is employed as PCR product in the productionmethod according to the invention. Likewise, the siRNA sequence can beproduced synthetically using oligodinucleotide synthesis. In this event,short ODN fragments can be used which must be annealed and ligated in afirst step, but it is also possible to produce the entire siRNA sequenceby means of ODN synthesis. The ODN fragment is phosphorylated by PNkinase.

The production process of the method according to the invention can bedescribed as follows and is illustrated in FIG. 1 for an overall view.

The plasmid bearing the promoter sequence is completely digested withthe BspTNI restriction enzyme at 37° C. overnight, thereby providing thepromoter fragment. Following addition of the respective siRNA sequenceand 5′-phosphorylated hairpin loop-shaped oligodeoxynucleotides indouble excess, the single fragments are ligated by means of the T4 DNAligase enzyme in the presence of the BspTNI restriction enzyme. Theresulting mixture of nucleic acids is treated with the T7 DNA polymeraseenzyme. The final product, i.e., the vector expressing siRNA, ispurified using column chromatography and is ready for transfection.

In one embodiment the vector expressing siRNA is envisaged to includetwo restriction sites allowing subsequent removal of the hairpins. Thisis advantageous in that the vector is available for further processes,be it cloning of the sequence into any desired expression vector, e.g. aplasmid, be it amplification of the sequence by PCR or the like. Thisembodiment is illustrated in FIG. 2.

An in vitro test was performed to check the functionality of the siRNAvectors according to the invention. To this end, hamster cells weretransfected with various siRNA vectors intended to suppress theexpression of luciferase. A plasmid and a vector according to theinvention were used as siRNA vectors. Both vectors achieved about 90%inhibition of luciferase expression. While having comparableeffectiveness and transfection efficiency, the method according to theinvention advantageously achieves production of a vector which avoidsthe above-described disadvantages of plasmid-based vectors and can beproduced in a much more time- and cost-saving fashion.

Hence, the significance of the invention lies in furnishing a method forthe production of suitable vectors which can be used in screeningprocedures, thus serving in rapid functional testing of potential siRNAsequences. Furthermore, the kit provides a potential tool for genetherapy in a sense that pathologic genes are switched off.

However, the production method also allows production of DNA expressionvectors in a simple manner. To this end, the siRNA sequence is replacedby a DNA sequence encoding a gene. Using restriction digestion, uniqueprotrusions are created at the ends of the DNA sequence, allowingligation of the fragments (promoter, polyA site and hairpin loop-shapedODN) in the proper arrangement. This pathway of production isschematically shown in FIG. 3. Also provided is a kit allowing theproduction of the DNA expression vectors. The components of the kits arethe following: a suitable plasmid with promoter and polyA sitesequences, coding DNA sequence, hairpin loop-shaped ODN, ATP, ligase,restriction enzyme, T7 polymerase, as well as column chromatographymaterial for the purification of the product.

Further advantageous measures are described in the supplementarysubclaims; the invention will be described in more detail with referenceto the examples and the following figures.

FIG. 1 shows the production pathway of the siRNA vectors.

-   -   A: siRNA sequence to be employed in the process, which is        homologous to the target mRNA. The sequence comprises a        sense-antisense-loop region and a termination sequence. The        siRNA sequence can be constituted of single ODN fragments which        must be annealed, ligated and optionally phosphorylated by means        of the enzyme mix, but it can also be present in the form of a        complete ODN fragment.    -   B: shows the components ligated to the ODN fragment by the        ligase enzyme. These components are the promoter sequence with        corresponding complementary protrusions and the hairpin        loop-shaped oligodinucleotides whose likewise complementary and        unique protrusions of 4 bases each result in the formation of a        covalently capped, linear vector which is constituted of the        promoter, sense, loop, antisense and termination sequences and        is capped at the ends in a hairpin loop shape.    -   C: Unligated components are degraded by T7 DNA polymerase        digestion in a final step. The remaining product is purified        using column chromatography.    -   D: Final product ready for transfection.

FIG. 2 shows the production pathway of the siRNA vectors with additionalrestriction sites.

-   -   A: siRNA sequence to be employed in the process, which is        homologous to the target mRNA. The sequence comprises a        sense-antisense-loop region and a termination sequence. The        siRNA sequence can be constituted of single ODN fragments which        must be annealed, ligated and optionally phosphorylated by means        of the enzyme mix, but it can also be present in the form of a        complete ODN fragment.    -   B: shows the components ligated to the ODN fragment by the        ligase enzyme. These components are the promoter sequence with        corresponding complementary protrusions, provided with an        additional restriction site at the 5′ end, and the hairpin        loop-shaped oligodinucleotides—a hairpin loop-shaped ODN        likewise bearing an additional restriction site—whose likewise        complementary and unique protrusions of 4 bases each result in        the formation of a covalently capped, linear vector which is        constituted of the promoter, sense, loop, antisense and        termination sequences and is capped at the ends in a hairpin        loop shape.    -   C: Unligated components are degraded by T7 DNA polymerase        digestion in a final step. The remaining product is purified        using column chromatography.    -   D: Final product ready for transfection, the product including        two restriction sites.

FIG. 3 shows the production pathway of coding DNA vectors.

-   -   A: A plasmid and a coding DNA sequence are used as starting        material. The plasmid bears restriction sites allowing excision        of the promoter and polyA site sequences.    -   B: The following fragments are formed as a result of restriction        digestion: promoter and polyA site, each having unique        complementary protrusions, and residual plasmid fragments. The        fragments are ligated after addition of coding DNA sequence,        hairpin loop-shaped ODN and in the presence of ligase enzyme.    -   C: Unligated components are degraded using T7 DNA polymerase        digestion. The covalently capped vector, constituted of        promoter, coding and polyA site sequences, is purified by means        of column chromatography.    -   D: DNA expressing vector usable in transfection.

FIG. 4 shows the in vitro inhibition of luciferase expression by siRNAExpression was determined following transfection of CHOK1 cells, usingrelative light units (rlu). The following was used: siRNA vectorproduced using the method according to the invention (a), plasmidbearing siRNA sequence (b), positive control to control the luciferaseexpression (c), untreated cells (d), and cells transfected with emptyvector (e). The values represent mean values calculated from a pluralityof determinations. In the negative and positive controls, suppression ofluciferase expression was not observed, as expected. In contrast, thesiRNA-treated cells showed significantly lower luciferase expression.Luciferase expression is reduced by up to 90% compared to the positivecontrol.

FIG. 5 shows the results of an experiment wherein the siRNA expressionvector according to the invention is compared with plasmids containingidentical expression cassettes.

-   -   CHO-K1 cells were cotransfected with 0.5 ng of plasmid encoding        Renilla luciferase, 4.5 ng of plasmid encoding firefly        luciferase, and 195 ng of the corresponding siRNA expression        construct directed against the expression of firefly luciferase.        The cells were lysed 24 hours after transfection, and the        luciferase activity was determined in a luminometer. The        activity of the firefly luciferase was balanced against the        activity of Renilla luciferase and compared with the activity of        the control (non-specific siRNA). The results illustrated show        the mean values of three independent tests.    -   The suppression of gene expression by MISECs produced with an        “siRNA Expression Vector Kit” using the method according to the        invention is comparable to the effects of plasmid transfections,        and the suppression of gene expression in both transfections        ranges between 70 and 75%.

FIG. 6 shows the dose dependence of gene repression followingtransfection of MISECs produced with an “siRNA Expression Vector Kit”,in which case the hairpin siRNA was directed against the fireflyluciferase gene, compared to non-specific siRNA sequences.

-   -   CHO-K1 were cotransfected with 500 ng of plasmid encoding        Renilla luciferase, 100 ng of plasmid encoding firefly        luciferase, and siRNA expression constructs against the firefly        luciferase gene in the specified quantities. The cells were        lysed after 24 hours, and the activity of the luciferases was        determined in a luminometer. The activity of the firefly        luciferase was balanced against the activity of Renilla        luciferase and compared with the activity of the control        (non-specific siRNA). The results illustrated show the mean        values of two independent tests.    -   Compared to the non-specific siRNA, transfection of the same        quantity (1000 ng) of MISECs produced with an “siRNA Expression        Vector Kit” using the method according to the invention shows a        significant decrease of the luciferase activity.

FIG. 7: In a further experiment carried out by the team of Dr.Christiane Kleuss at the Institut für Pharmakologie der FreienUniversität Berlin, the effectiveness of gene repression by MISECsproduced with an “siRNA Expression Vector Kit” using the methodaccording to the invention was compared to that of a plasmid containingthe same expression cassette.

-   -   CHO-K1 were cotransfected with 12 ng of plasmid encoding Renilla        luciferase, 6 ng of plasmid encoding firefly luciferase, and 182        ng of a corresponding siRNA expression construct against the        firefly luciferase gene. CHO-K1 were cotransfected with 500 ng        of plasmid encoding Renilla luciferase, 100 ng of plasmid        encoding firefly luciferase, and siRNA expression constructs        against the firefly luciferase gene in the specified quantities.    -   The cells were lysed after 24 hours, and the activity of the        luciferases was determined in a luminometer. The activity of the        firefly luciferase was balanced against the activity of Renilla        luciferase and compared with the activity of the control        (non-specific siRNA). The results illustrated show the mean        values of three independent tests.    -   In this experiment as well, the siRNA expression vectors        produced according to the method of the invention show the same        effectiveness as the plasmid with identical expression cassette,        each time being about 75% reduction of the luciferase activity        compared to the non-specific control plasmid. The inhibition of        the constructs produced according to the invention is absolutely        sufficient for effective identification of successful target        sequences in a screening procedure within a short time, which        are suitable for gene repression.

EXAMPLE 1 Production of siRNA Vectors for the Suppression of LuciferaseExpression

The vectors encoding the siRNA of luciferase (siRNALuc) were obtained asfollows:

The two ODN fragments for siRNALuc were heated at 90° C. for 3 min andannealed by slow cooling. In this way, the following sequence encodingluciferase was obtained:

SEQ ID NO. 1: GAGCTGTTTC TGAGGAGCCT TCAAGAGAGG CTCCTCAGAA ACAGCTC

Therein, the first 19 bases constitute the sense strand, the following 9bases the loop region, and the remaining 19 bases the antisense strand.

Phosphorylation by means of PN kinase was effected subsequently. Toobtain 10 micrograms of final product, an amount of 3.9 micrograms ofsiRNALuc was used. Following addition of 5.2 micrograms of H1 promoter(SEQ ID NO. 2) and of 5′-phosphorylated hairpin loop-shapedoligodeoxynucleotides (SEQ ID NO. 3 and 4):

SEQ ID NO. 2: ATATTTGCAT GTCGCTATGT GTTCTGGGAA ATCACCATAA ACGTGAAATGTCTTTGGATT TGGGAATCTT ATAAGTTCTGT ATGAGAGCAC AGATAGGG SEQ ID NO. 3:5′-PH-GGG AGT CCA GTT TTC TGG AC-3′ (1.2 μg) and SEQ ID NO. 4: 5′-PH-TGGAAA GTC CAG TTT TCT GGA CTT-3′ (1.4 μg),the individual fragments were ligated using the T4 DNA ligase enzyme inthe presence of the BspTNI restriction enzyme. The resulting mixture ofnucleic acids was treated with the enzyme T7 DNA polymerase. The finalproduct, i.e., the vector expressing siRNALuc, was purified by columnchromatography and was ready for transfection.

EXAMPLE 2 Suppression of Luciferase Expression In Vitro

Hamster cells of the CHOK1 cell line were seeded in 24-well plates insuch a way that 8×10⁴ cells in 500 μl of medium were seeded per well.Following incubation over 24 h, transfection with various constructsexpressing siRNALuc was effected. FuGene6 was used as transfectionreagent. As reference vector for the determination of the fireflyluciferase activity, a plasmid encoding Renilla luciferase was used ineach batch in addition to plasmid encoding firefly luciferase. In thisway, the firefly luciferase expression in relation to the marker Renillaluciferase expression can be determined by means of a dual assay.Non-transfected cells and cells transfected with a blank vector wereused as negative controls. After incubation overnight, the cells werelysed and taken up in 15 μl of passive lysis buffer each time.Expression was detected using a dual luciferase reporter assay in aluminometer. The result is illustrated in FIG. 4.

EXAMPLE 3 Production of Coding DNA Vectors Using the Method According tothe Invention

DNA vector production is effected in a combined restriction/ligationbatch. The pMCV2.8 plasmid being used has four BspTNI and Eco31Irestriction sites, providing promoter and polyA site after digestion.

The plasmid and the coding DNA fragment are digested with the BspTNIrestriction enzyme and ligated with the hairpin loop-shaped ODNs.Sequences of the hairpin loop-shaped ODNs:

SEQ ID NO. 3: 5′-PH-GGG AGT CCA GTT TTC TGG AC-3′ and SEQ ID NO. 5:5′-PH-AGG GGT CCA GTT TTC TGG AC-3′.

Thus, the restriction/ligation batch includes: plasmid, coding DNAsequence, hairpin loop-shaped ODN, reaction buffer, ATP, BspTNI and T4DNA ligase. Incubation is performed over 4 h at 37° C. The process isquenched by heat inactivation for 15 min at 70° C.

Degradation of residual vector and any non-ligated fragments is effectedusing T7 DNA polymerase digestion. The coding vector is purified bymeans of chromatography and is ready for transfection.

1. A method for producing vectors which, following transfection thereof into eukaryotic cells, specifically inhibit formation of defined proteins therein by RNA interference, said method comprising: a) mixing a DNA double strand which comprises a singular copy, 19-23 bases in length, of a gene sequence, once in 5′-3′ direction and once in 3′-5′ direction, a sequence, 8-12 bases in length, of two DNA single strands each being arranged between the 5′-3′ and 3′-5′ oriented singular copy of the gene sequence, said single strands being selected such that opposite bases are by no means complementary to each other and double strand regions flanking them are linked to each other by said two DNA single strands, said DNA double strand having short protruding ends of single-stranded DNA at its ends, with hairpin loop-shaped oligodeoxynucleotides having short protruding ends of single-stranded DNA at their ends, and a promoter having short protruding ends of single-stranded DNA, a single-stranded 5′ end of the promoter being capable of pairing with one of the hairpin loop-shaped oligodeoxynucleotides, and a single-stranded 3′ end of the promoter being complementary to a single-stranded 5′end of the DNA double strand, and a termination signal for RNA polymerases with short protruding ends of single-stranded DNA, a 5′ protrusion of the termination signal being capable of specific pairing with a 3′ end of the DNA double strand, and a 3′ protrusion of the termination signal being capable of specific pairing with a hairpin loop-shaped oligodeoxynucleotide, b) subsequent ligation of the DNA fragments, and c) final purification of the vectors produced.
 2. The method according to claim 1, wherein the promoter is part of a bacterially amplifyable plasmid which, prior to mixing the components in 1a), is cut with a restriction endonuclease recognizing a restriction site flanking the promoter on the plasmid, wherein the restriction site is not present on the molecule to be produced.
 3. The method according to claim 2, wherein the ligation step according to 1 b) is effected in presence of the restriction endonuclease by means of which the promoter has been excised from the plasmid.
 4. The method according to claim 2 or 3, wherein the step of final purification according to 1 c) is preceded by digestion of the reaction mixture, using an exonuclease specific for 3′ or 5′ DNA ends only.
 5. The method according to at least one of claims 2 or 3, wherein the restriction endonuclease is an enzyme from the group of class II restriction endonucleases, preferably an enzyme from the group of BbsI, BbvI, BbvlI, BpiI; BplI, BsaI, BsmAI, BsmBI, BsmFI, BspMI, Eam104I, Earl, Eco31I, Esp3I, FokI, HgaI, SfaNI or isoschizomers thereof.
 6. The method according to claim 1, wherein the mixture from 1 a) is added with a DNA double strand resulting from partial annealing of a partially self-complementary oligodeoxynucleotide or of at least two oligodeoxynucleotides.
 7. The method according to claim 1, wherein the promoter being added is the promoter of the human gene for H1 RNA (SEQ ID NO. 2).
 8. The method according to claim 1, wherein the hairpin loop-shaped oligodeoxynucleotides have a recognition sequence for a restriction endonuclease in their double-stranded region.
 9. The method according to claim 1, wherein the purification in 1 c) is effected using chromatography and/or gel electrophoresis.
 10. A kit comprising at least one promoter, hairpin loop-shaped oligodeoxynucleotides, and enzymes for production of vectors according to claim 1 which, following their transfection into eukaryotic cells, are suitable for targeted inhibiting formation of defined proteins therein by RNA interference.
 11. The kit according to claim 10, wherein said enzymes are selected from restriction endonucleases, restriction exonucleases, ligases, kinases and polymerases.
 12. The kit according to claim 10 or 11, wherein said kit additionally comprises means for-performing the enzymatic reactions.
 13. The kit according to claim 10 or 11, wherein said kit additionally comprises means for purifying the vectors produced.
 14. The kit according to claim 10, wherein the promoter is included as part of a bacterially amplifyable plasmid.
 15. The kit according to claim 10, wherein said kit comprises a restriction endonuclease suitable for excision of the promoter from the plasmid.
 16. A vector which, following transfection in eukaryotic cells, specifically inhibits formation of defined proteins by RNA interference, wherein said vector is capped by hairpin loop-shaped oligodeoxynucleotides having arranged therebetween a promoter at a 5′ end and a termination signal at a 3′ end of a DNA double strand, said DNA double strand comprising a singular copy, 19-23 bases in length, of a gene sequence, once in 5′-3′ direction and once in 3′-5′ direction, a sequence 8-12 bases in length of two single strands each being arranged between the 5′-3′ and 3′-5′ oriented singular copy of the gene sequence, said single strands being selected such that opposite bases are by no means complementary to each other and double strand regions flanking them are linked to each other by two DNA single strands. 